1. Field of the Invention
The present invention relates to a technique for preparing an insect culture cell system.
2. Prior Art
There are two stages in the preparation of an animal culture cell line including that of insects. The initial stage is called primary culture, and the next stage is called subculture. When a culture cell line is prepared, a target tissue is extracted from an insect of interest in an aseptic manner. The tissue is then put into a culturing flask together with a cell culture medium, and cultured for about one year, until cells emerge from the tissue section onto the flask surface and divide into sufficient numbers. The sufficiently multiplied cells are transplanted into a new flask where they are subjected to subculture, i.e., culturing of a prepared cell line.
In the prior art, a variety of insect cell media are available. Examples include Grace's media, IPL-41 media, Schneider's Drosophila media, Sf900II, TC-100 media, Sf-9 cell media, Sf-21 cell media, Express Five media, and EX-400 media. Mitsuhashi, J. et al. have prepared a medium along the line of MGM, for example, by referring to the results of insect body fluid analysis (MGM-443; Mitsuhashi, J. (1980) In: Kurstak, E., Maramorosch, K. and Dubendorfer, A. (eds) Invertebrate Systems In Vitro Elsevier/North Holland Biomedical, Amsterdam, pp. 47–58, MGM-448; Mitsuhashi, J. (1984) Zool. Sci. 1, pp. 415–419, MGM-450; Mitsuhashi, J. and Inoue, H (1988) Appl. Entomol. Zool. 23, pp. 488–490, MGM-464; Mitsuhashi, J. (2001) In Vitro Cell. Dev. Biol. 37A, pp. 330–337. MGM-443, MGM-448, MGM-450, and MGM-464 are media names.).
The present inventors have prepared MM-8 SF medium, for example (Shigeo Imanishi, “Konchu Kino Jikken-kei Oyobi Konchu Saibo Baiyo-kei No Kaihatsu: Kenkyu Seika 295” (“Development of Insect Function -Experiment System and Insect Cell Culture System: Study Results 295”), ed. by Agriculture, Forestry, and Fisheries Research Council, pp. 74–84 (1994)). These media, however, are not suitable for primary culture, and are used mostly for the subcultivation of a prepared culture cell line, i.e., for the maintenance of the cells after development of the cell line. When these media were used for primary culture, cell growth was not satisfactory, and primary culture took as long as about one year, as mentioned above. Thus, there has been no medium suitable for primary culture which enables the cells to be transferred into subculture at an early stage. Moreover, many of these media were limited for use with certain insect species. For example, the Schneider's Drosophila medium was for the culturing of cells of insects of the order Diptera, particularly fruit flies, while Sf900II, Sf-9 cell medium and Sf-21 cell medium were for the cells of insects of the order Lepidoptera, particularly inch worms. Thus, there has so far been no medium suitable for the growth of cells of insects of a wide variety of orders.
On the other hand, flasks generally used for cell culture are made of plastic, and are believed to have a coating of extracellular matrix on the plastic surface, such as, e.g., collagen I, II, III, IV, or V, fibronectin, gelatin, laminin, poly-L-lysine, Matrigel, and EHS-matrix. There are not many detailed reports about their compositions, but as to chitin and chitosan, there have been reports about an extracellular matrix extracted from crustaceans such as lobsters and crabs. These chitin and chitosan have been variously chemically modified. The extracellular matrix provides the effect of attaching a tissue to the cell culture vessel, and adhering those cells that have transmigrated from the tissue to the vessel surface for division and multiplication. However, there is a need for an extracellular matrix with a higher cell adhesion ability for good cell culture.